In vitro analyses of mitochondrial ATP/ phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Lorenz et al. BMC Plant Biology (2015) 15:238
DOI 10.1186/s12870-015-0616-0

RESEARCH ARTICLE

Open Access

In vitro analyses of mitochondrial ATP/
phosphate carriers from Arabidopsis
thaliana revealed unexpected Ca2+-effects
André Lorenz1, Melanie Lorenz1, Ute C. Vothknecht2, Sandra Niopek-Witz3, H. Ekkehard Neuhaus3
and Ilka Haferkamp1*

Abstract
Background: Adenine nucleotide/phosphate carriers (APCs) from mammals and yeast are commonly known to
adapt the mitochondrial adenine nucleotide pool in accordance to cellular demands. They catalyze adenine
nucleotide - particularly ATP-Mg - and phosphate exchange and their activity is regulated by calcium. Our current
knowledge about corresponding proteins from plants is comparably limited. Recently, the three putative APCs from
Arabidopsis thaliana were shown to restore the specific growth phenotype of APC yeast loss-of-function mutants
and to interact with calcium via their N-terminal EF-hand motifs in vitro. In this study, we performed biochemical
characterization of all three APC isoforms from A. thaliana to gain further insights into their functional properties.
Results: Recombinant plant APCs were functionally reconstituted into liposomes and their biochemical characteristics
were determined by transport measurements using radiolabeled substrates. All three plant APCs were capable of ATP,
ADP and phosphate exchange, however, high preference for ATP-Mg, as shown for orthologous carriers, was not
detectable. By contrast, the obtained data suggest that in the liposomal system the plant APCs rather favor ATP-Ca as
substrate. Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects
on ATP transport did not primarily/essentially involve Ca2+-binding to the EF-hand motifs in the N-terminal domain of
the carrier.
Conclusion: Biochemical characteristics suggest that plant APCs can mediate net transport of adenine nucleotides and
hence, like their pendants from animals and yeast, might be involved in the alteration of the mitochondrial adenine
nucleotide pool. Although, ATP-Ca was identified as an apparent import substrate of plant APCs in vitro it is arguable

whether ATP-Ca formation and thus the corresponding transport can take place in vivo.
Keywords: Mitochondria, calcium, Ca2+, Signaling, Energy, Adenine nucleotide transport, Plant, ATP, ADP, Phosphate

Background
The mitochondrial carrier family (MCF) comprises
structurally related but functionally diverse proteins
that are characteristic for and generally restricted to eukaryotes [1–5]. MCF proteins represent the main solute
carriers in the inner mitochondrial membrane and
catalyze the translocation of various metabolites, such
as nucleotides, cofactors, carboxylates, amino acids etc
(for review see [6]).
* Correspondence:
1
Cellular Physiology/Membrane Transport, University of Kaiserslautern, 67653
Kaiserslautern, Germany
Full list of author information is available at the end of the article

Mitochondrial ATP-Mg/phosphate carriers (APCs)
represent a specific MCF subgroup comprising carriers
from different eukaryotes that are phylogenetically related
to the well characterized ADP/ATP carriers (AACs) required for mitochondrial energy passage (for review see
[6, 7]). Over the past years the physiological and biochemical properties of the single yeast APC isoform Sal1p (suppressor of Δaac2 lethality) as well as of various
mammalian homologs became more and more clarified
[8]. Initially, Sal1p was shown to suppress the growth
phenotype of yeast impaired in mitochondrial energy
transport (due to AAC deletion or inhibition). In a similar
fashion, AAC compensates the loss of functional Sal1p

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Lorenz et al. BMC Plant Biology (2015) 15:238

[8]. Subsequent studies revealed that Sal1p and its mammalian homologs mediate the counter exchange of adenine nucleotides and phosphate [9–13]. Therefore, the
redundant physiological function of Sal1p and AAC supposedly was not primarily energy exchange but adenine
nucleotide translocation, most likely ATP entry into mitochondria [13, 14].
Alteration of the mitochondrial adenine nucleotide
pool by adenine nucleotide exchange with phosphate was
shown to affect different physiological processes, such as
glucose metabolism, oxidative phosphorylation, mitochondrial biogenesis and DNA maintenance in yeast or mammals [9–13]. APC proteins apparently prefer two-fold
negatively charged substrates, either ATP in complex with
Mg2+ (ATP-Mg2−), protonated ADP (HADP2−) or HPO2−
4 ,
which makes the catalyzed transport electroneutral [15].
The composition (respective concentrations) of the different substrates at the matrix and cytosolic sides of the carrier determine whether adenine nucleotides preferentially
become exported or imported [11, 15].
Interestingly, addition of Ca2+ to isolated mitochondria
as well as metabolic situations that result in increase of
free cytosolic Ca2+ were shown to enhance mitochondrial adenine nucleotide levels by stimulation of APC activity in mammals and yeast [8, 16–18] (for review see
[19]). In one aspect APC proteins considerably differ
structurally from typical MCF proteins; they are Nterminally extended by a domain that is exposed to the
inter-membrane space of the mitochondrion and contains up to four putative Ca2+-binding EF-hand motifs
[20–22]. Very recent structural studies with the Nterminal domain of human APC isoform 1 (also termed
SCaMC1 for short Ca2+-dependent mitochondrial Carrier 1) showed that the Ca2+-bound state is quite compact and rigid whereas the apo (Ca2+-free) state
appeared more flexible [21, 22]. Moreover, interaction
studies with the two individual SCaMC1 domains, the

Ca2+-binding part and the C-terminal transmembrane
region, led to the assumption that the apo state of the
N-terminal domain forms a cap that closes the translocation pathway whereas Ca2+-binding causes cap removal/opening and thus transporter activation [21, 22].
In contrast to yeast and mammals [8, 12, 16, 18, 23–25]
analyses concerning the net adenine nucleotide transport
of mitochondria in plants are still rudimentary. Previous
studies led to controversial results but have indicated that
plant mitochondria are capable of net adenine nucleotide
uptake [26–31]. Arabidopsis thaliana possesses three putative APC proteins (AtAPC1-3) that exhibit high amino
acid sequence similarities to their human and yeast counterparts. Phylogenetic analysis of MCF proteins shows that
APCs cluster together and that plant APCs form a sister
group to the human and yeast orthologs [6]. Similar to
yeast or mammalian APCs, the plant pendants contain an

Page 2 of 16

N-terminal extension with four putative EF-hand motifs
and were recently shown to interact with Ca2+ at least
in vitro [32]. Moreover, all three plant isoforms were
able to rescue the specific growth phenotype of Δsal1p
yeast mutants [32]. Therefore, AtAPC1-3 isoforms were
suggested to represent Ca2+-regulated ATP-Mg/phosphate transporters. To gain first insights into the biochemical characteristics of the three APCs from A.
thaliana we reconstituted the heterologously expressed
proteins into liposomes and investigated their capacity
for adenine nucleotide transport. Our data indicate that
plant APCs mediate antiport of ATP, ADP and phosphate and therefore might be involved the alteration of
the mitochondrial adenine nucleotide pool. Moreover,
the determined transport characteristics suggest that in
the in vitro system, the plant APCs preferentially import
the Ca2+- and not the Mg2+-complexed form of ATP.

Methods
Generation of expression constructs

The coding sequences of AtAPC1-3 were amplified
from Arabidopsis cDNA with specific primers via Pfupolymerase-mediated PCR. For generation of the truncated AtAPC2 mutant protein lacking its Ca2+-interacting N-terminus a sense primer was chosen that
internally hybridizes with the corresponding full-length
sequence resulting in a recombinant protein starting at
amino acid position 164 directly after the fourth predicted EF-hand motif coding region. The isopropyl
β-D-thiogalactopyranoside (IPTG)-inducible T7 RNA
polymerase pET-vector/Rosetta™ 2 expression system
(Merck Biosciences, Novagen®, Darmstadt, Germany)
was used for heterologous protein synthesis. Accordingly, the primers were adapted to allow insertion into
the expression vector pET16b in frame with the
histidine-tag coding sequence. The coding sequence of
AtAPC1 was inserted via NdeI (sense primer) and XhoI
(antisense primer) whereas the remaining sequences
were inserted via XhoI (sense primer) and BamHI
(antisense primer). Correctness of the respective expression constructs was verified by sequencing.
Heterologous protein synthesis and detection

For heterologous protein synthesis Rosetta™ 2 cells were
transformed with the expression constructs and cultured
in 50 mL standard Terrific Broth (TB) medium at 37 °C
under vigorous shaking. At an OD600 of 0.5, expression
was induced by addition of 1 mM IPTG. Two hours
after induction, cells were concentrated by centrifugation
(3000 g, 5 min, 4 °C) and rapidly frozen (in liquid nitrogen). The frozen cell pellet was resuspended in buffer R
(25 % sucrose, 50 mM Tris, pH 7.0, 1.5 % Triton X-100,
18.75 mM EDTA) supplemented with 1 mM PMSF, a

pinch of DNAse and RNAse and incubated for


Lorenz et al. BMC Plant Biology (2015) 15:238

approximately 30 min at 37 °C to stimulate autolysis by
the endogenous lysozyme which was released from the
cells due to the freeze/thaw procedure. Subsequent sonication additionally supported cell disruption. Inclusion
bodies were separated from soluble and membrane proteins of the cell homogenate by centrifugation (20,000 g,
15 min, 4 °C).
For documentation of heterologous protein synthesis,
an aliquot of the inclusion bodies fraction was used for
SDS-PAGE, Western-blotting and immune detection.
For this, inclusion bodies were resuspended in buffer R
and an appropriate volume of 6 x concentrated sample
buffer medium (375 mM Tris/HCl, pH 6.8, 0.3 % SDS,
60 % glycerol, 1.5 % bromophenol blue) was added. Protein separation was performed in a discontinuous, denaturing system with a 3 % stacking and a 12 % separating
polyacrylamide gel [33]. Following electrophoresis, the gel
was coomassie stained or used for Western-blotting. Immune detection was performed using a monoclonal anti
poly His IgG (Sigma; ) combined with a secondary alkaline phosphatase conjugated
anti-mouse IgG (Sigma). Alkaline phosphatase activity
was detected by staining with nitro blue tetrazolium chloride/5-bromo-4-chloro-3’-indoly phosphate toluidine salt.

Page 3 of 16

PIPES, pH 7.0, 20 mg phosphatidylcholine, 1.6 mg cardiolipin, 28 mg C10E5) and detergent removal by
amberlite XAD-2 beads was performed exactly as given
by Heimpel et al., [34]. Overnight incubation with biobeads completed protein refolding and proteoliposome
formation. External buffer medium and loading substrates
were removed from the vesicles (500 μL) by desalting with

NAP-5 columns (GE Healthcare; ealthcare.
com). Columns were equilibrated and liposomes were
eluted with of import buffer (50 mM NaCl, 10 mM PIPES,
pH 7.5). For transport measurements 50 μL of these proteoliposomes were mixed with 50 μL of import buffer supplemented with the indicated concentrations of [α32P]ATP, [α32P]-ADP, [45Ca], MgCl2 and CaCl2 and incubated
at 30 °C. At the given time points import was terminated
by removal of external import medium via vacuum filtration as described in [35]. Briefly, liposomes were loaded to
pre-wetted filters (mixed cellulose ester, 0.45-μm pore size;
Whatman) and washed rapidly with phosphate buffer.
Imported radioactivity was quantified by scintillation
counting (Beckman LS6500; Beckman Coulter). For [45Ca]
uptake measurements import was terminated and nonimported Ca2+ was removed by EGTA addition (2 mM)
and incubation for 15 s prior to vacuum filtration and
washing.

Purification of inclusion bodies

Basically, purification of inclusion bodies as well as their
solubiliztaion, refolding and integration into lipid/detergent micelles was performed according to [34]. For this,
the cell pellet of the inclusion body fraction washed in
buffer W1 (20 ml 1 M urea, 1 % Triton X-100 and 0.1 %
β-mercapto-ethanol). After centrifugation (20,000 g,
15 min, 4 °C) inclusion bodies were additionally washed
in buffer W2 (20 mM Tris, pH 7.0, 0.5 % Triton X-100,
1 mM EDTA, 0.1 % β-mercapto-ethanol) and finally in
buffer W3 (50 mM Tris, pH 7.0, 1 mM EDTA, 0.1 % βmercapto-ethanol). Solubilization of the purified inclusion body proteins was achieved by resuspension in buffer medium S (10 mM Tris, pH 7.0, 0.1 mM EDTA,
1 mM DTT, 0.05 % polyethylene glycol 4000) containing
1.67 % of the detergent n-lauroylsarcosine and incubation for 15 min on ice. The protein fraction was diluted
(threefold) with 10 mM Tris (pH 7.0) and finally, the
solubilized proteins were separated from insoluble aggregates by centrifugation (12,000 g, 4 min, 4 °C).
Preparation of proteoliposomes and transport

measurements

For preparation of proteoliposomes 100 μg of the solubilized proteins were mixed with 20 mM Hepes, pH 7.0
and 1 mM PMSF. To obtain vesicles with internal
counter exchange substrates 5 mM of phosphate or adenine nucleotides were added to the protein mixture.
Preparation of mixed detergent-lipid micelles (100 mM

Results
Recombinant plant APCs act as ATP, ADP and Pi antiporters

To determine functional properties of the different APCs
from A. thaliana we used the heterologous Escherichia
coli expression system for production of the respective
isoforms and performed transport measurements after carrier reconstitution into artificial lipid vesicles, so called liposomes. This approach was previously successfully applied
to biochemically characterize several MCF proteins, including two selected human SCaMC isoforms [12, 34, 36–38].
The three plant APCs were heterologously expressed
as N-terminal His-tag fusions. Like previously observed
for many MCF proteins [12, 34, 36–38] also plant APCs
were synthesized at high levels and accumulated in form
of insoluble inclusion bodies (Additional file 1: Figure
S1A and B). The aggregated proteins were enriched,
purified, solubilized and finally refolded during their integration into liposomes.
Import measurements were performed on proteoliposomes either harboring or lacking selected possible
counter exchange substrates in the lumen (Fig. 1,
Additional file 2: Figure S2). This allowed investigation of in vitro transport activities and hence functionality of the reconstituted proteins as well as of the
catalyzed transport mode. All recombinant plant
APCs mediated time dependent uptake of [α32P]-ATP
into phosphate (Pi) loaded liposomes (Fig. 1a, c, e,
black rhombs) and no comparable accumulation of

Lorenz et al. BMC Plant Biology (2015) 15:238

Page 4 of 16

Fig. 1 Time dependent ATP transport via AtAPC1-3. Transport of 50 μM [α32P]-ATP into Pi (a, c, e) and into ATP (b, d, f) loaded proteoliposomes with
reconstituted AtAPC1 (a, b), AtAPC2 (c, d) and AtAPC3 (e, f). ATP uptake was measured in absence (black rhombs) and presence (gray circles) of 500 μM
externally applied MgCl2. Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of radioactivity and the
corresponding rates were unaffected by MgCl2 addition. Data represent mean values of at least three independent replicates, standard errors are given

radioactivity was observable with corresponding vesicles lacking Pi in the lumen (Fig. 1a, c, e, open
rhombs). This observation already demonstrates that
plant APCs can act as antiporters; ATP/Pi exchange
by the different APC isoforms was linear for at least
5 min. Maximal uptake via AtAPC1 of ~ 6 nmol/mg protein was reached after 10 to 15 min (Fig. 1a, black rhombs),
whereas AtAPC2 and AtAPC3 show marginally or considerably higher transport rates that approached a maximum
of ~ 9 nmol/mg protein and ≥ 17 nmol/mg protein after
20 min, respectively (Fig. 1c and e, black rhombs).
Yeast Sal1p and mammalian SCaMCs were shown to
discriminate against free ATP as substrate or at least to
prefer the Mg2+-complexed form of ATP over free ATP

[12, 15, 16, 39]. To check whether this is also true for
the plant APCs, the influence of Mg2+ on ATP transport
was analyzed. To this end, the ATP transport medium was
supplemented with 500 μM Mg2+ to convert ~ 80 % of free
ATP (ATP4−) into the Mg2+-complexed form (ATP-Mg2−)
( />[40]). ATP-Mg2− and HPO2−
4 exchange results in an electroneutral transport. In case of AtAPC1 and AtAPC3
addition of Mg2+ caused moderate (~1.6-fold to 2.0-fold)

increase in adenine nucleotide/Pi exchange compared to
ATP without Mg2+ (Table 1; Fig. 1a and e, compare gray
circles and black rhombs) whereas transport by AtAPC2
was stimulated to a lesser extent (Table 1; Fig. 1c, compare
gray circles and black rhombs).


Lorenz et al. BMC Plant Biology (2015) 15:238

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Table 1 Comparison of counter exchange rates of AtAPC1-3
Exchange (import/export)
ATP/Pi
ATP-Mg/Pi

AtAPC1

AtAPC2

AtAPC3

4.1

5.0

7.2

6.4

6.7

13.5

ATP/ATP

17.4

7.7

20.1

ATP-Mg/ATP

28.9

10.3

28.1

7.6

5.7

11.3

39.8

12.2

42.0

ADP/Pi
ADP/ADP

ATP and ADP transport was allowed for 10 min. Rates represent net values of
transport (minus corresponding transport into non-loaded liposomes) and are
given in nmol/mg protein. For investigation of Mg2+ impact on ATP uptake
500 μM of MgCl2 were added to the transport medium. The complete time
courses of ATP and ADP transport are displayed in Fig. 1 and in Additional file
2: Figure S2

To unravel whether the stimulatory influence of Mg2+
on ATP uptake is due to general preference for ATPMg as substrate or rather due to the electroneutrality
of the corresponding transport process we investigated
Mg2+-effects on ATP homo-exchange. Homo-exchange
of ATP is electroneutral but becomes electrogenic
when ATP-Mg2− is exchanged with ATP4−. Comparison
of the transport rates indicates that AtAPC1 highly,
AtAPC3 markedly and AtAPC2 slightly prefer ATP
homo-exchanges over the corresponding ATP/Pi heteroexchanges (Table 1; compare Fig. 1a, c, e with b, d, f, black
rhombs). Moreover, ATP homo-exchanges of all three
AtAPCs became further enhanced by Mg2+ (Fig. 1b, d, f,
compare gray circles and black rhombs) and the degree of
Mg2+-dependent stimulation was nearly identical to that
of the ATP/Pi hetero-exchange (Table 1). The observed
stimulatory effects of Mg2+ on ATP/Pi and ATP/ATP
transport indicate that AtAPC1 and 3 generally prefer
ATP-Mg as substrate whereas AtAPC2 apparently only
slightly favors the Mg2+-complexed form.

Because ADP represents an additional substrate of
yeast Sal1p and human SCaMCs [12, 15, 18, 39] we verified whether this nucleotide is also transported by the
plant orthologs in our in vitro system. For this, uptake
of radiolabeled ADP into differentially loaded liposomes
was measured. All plant APCs transported ADP in
hetero-exchange with Pi as well as in homo-exchange
with ADP and no import occurred into non-loaded vesicles (Additional file 2: Figure S2). The rates of ADP
transport (in exchange with Pi or ADP) largely resemble
the rates of the corresponding Mg2+-stimulated ATP
transport (in exchange with Pi or ATP) (Table 1; compare Additional file 2: Figure S2 and Fig. 1). Just like observed for ATP transport, all plant APCs favor the
homo-exchange of ADP over the corresponding ADP/Pi
hetero-exchange and this preference is highly pronounced for AtAPC1 followed by AtAPC3 and finally
AtAPC2 (Table 1). Moreover, comparison of the rates of
ATP and ADP homo-exchanges with those of the

corresponding Pi hetero-exchanges (Table 1) suggests
that AtAPC2 in contrast to AtAPC1 and 3 does not
strongly discriminate between nucleotides or Pi as internal counter exchange substrate. The ineffectiveness of
non-loaded vesicles to induce significant import of ATP
or ADP also demonstrates that vesicles do not allow
carrier-independent passage of the labeled compounds.

Calcium differentially affects ATP and ADP transport
properties of the plant APCs

Diverse physiological data indicate a Ca2+-dependent
regulation of mitochondrial net adenine nucleotide passage [16–18, 39, 41]. In the native environment many
factors such as activity of adenylate kinases, Ca2+-induced metabolic processes, the mitochondrial membrane potential, respiration, Mg2+ complexation of ATP,
etc. influence internal and external adenylate and Pi
pools and consequently also mitochondrial adenine nucleotide translocation in general [42–45].

Transport studies with reconstituted APCs might provide a suitable tool to overcome interfering metabolic and
physiological effects and to study the impact of Ca2+on
this process in more detail. However, it is important to
mention that transport of reconstituted human SCaMC1
was not stimulated by Ca2+ addition [12] and also
AtAPC1-3 are already active in the absence of any Ca2+
addition (Fig. 1 and Additional file 2: Figure S2). These
findings suggest that Ca2+ is not essentially required for
carrier activation or that Ca2+contaminations exist in the
buffer media. Determination of cations (by ion chromatography) revealed that in fact traces of both, Ca2+ and
Mg2+, are present in the media (~9 μM, respectively).
If Ca2+ is essential for carrier activation and under the
assumption that the proteoliposomes still contain a certain amount of inactive (Ca2+-free) APC proteins, an
addition of extra Ca2+ should result in transport stimulation. To investigate a possible Ca2+-induced increase in
transport activation we performed uptake studies with
and without 200 μM Ca2+. Elevated Ca2+ availability generally stimulated nucleotide uptake of all three plant APCs
(Table 2). This observation might point to a Ca2+-induced
activation of previously inactive (Ca2+-free) carrier proteins. Studies with the two separately expressed subdomains (N-terminal domain and membrane spanning
part) of the human SCaMC1 led to the conclusion that
the N-terminal domain acts as a lid that either opens or
closes the translocation pathway in response to Ca2+ availability [22]. Given that Ca2+ exclusively causes removal of
the N-terminal domain and hence activation of previously
closed carriers, the same degree of stimulation would be
expected independent of the kind of substrate exchanged.
However, direct comparison of Ca2+ influence on different
exchanges shows that for the reconstituted plant APCs


Lorenz et al. BMC Plant Biology (2015) 15:238

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The different effects of Ca2+ on ATP and ADP
transport properties indicate that besides its proposed function in cap removal and carrier activation
Ca2+ fulfills an additional role in substrate transport/
recognition.

Table 2 Stimulation of the given exchanges by addition of
200 μM CaCl2
Exchange (import/export)

AtAPC1

AtAPC2

AtAPC3

ATP/Pi

3.05

3.12

4.30

ATP-Mg/Pi

1.67

2.75

1.94

ATP/ATP

2.65

3.39

3.62

ATP-Mg/ATP

1.59

2.37

1.82

ADP/Pi

1.55

2.03

1.49

ADP/ADP

1.56

2.12

1.54

ADP/ATP

1.79

2.20

1.70

Ca2+ effects override Mg2+ effects on ATP transport

To approach the function of Ca2+ during plant APC mediated transport it is important to keep in mind that
ATP can form a complex with Mg2+as well as with Ca2+
and it is thus imaginable that plant APCs are capable of
ATP-Ca transport in vitro.
Comparison of Ca2+ effects on ATP and ATP-Mg
transport indeed revealed interesting results that support
this assumption. Mg2+ addition causes marginal (AtAPC2)
to moderate (AtAPC1 and 3) increase in ATP transport
when no extra Ca2+ is present (Figs. 1 and 2). With rising
Ca2+ concentration the positive impact of Mg2+ becomes
abolished and even reverted into a negative one (Fig. 2).
More precisely, with higher Ca2+ concentrations (>10 μM
AtAPC2; > 50 μM AtAPC1 and 3) the rates of ATP transport in absence of Mg2+ exceed the rates of the corresponding exchange in presence of Mg2+. Accordingly, in
presence of Mg2+ higher concentrations of Ca2+ are apparently required to achieve ATP-transport saturation.

Ca2+ −dependent stimulation (x-fold) was calculated according to
corresponding transport in absence of Ca2+. SE are always below 15 % of the
given value

the degree of stimulation is higher for ATP than for ADP
or ATP-Mg uptake (Table 2).
We thus determined the apparent biochemical parameters of ATP/Pi and ADP/ATP exchange for all
three AtAPCs in more detail (Table 3). Velocity of
transport of all recombinant carriers approached saturation with increasing ATP or ADP concentrations
and conformed to simple Michaelis-Menten kinetics
(Additional file 3: Figure S3). The individual AtAPC
isoforms differed in their respective ADP affinities
(AtAPC1: 180 μM, AtAPC2: 374 μM and AtAPC3:
72 μM) whereas the ATP affinities were more similar
(ranging from 68 to 113 μM). Affinities of AtAPC1 for
ATP and ADP remained more or less unaffected by
Ca2+ addition whereas ATP affinities of AtAPC2 and
AtAPC3 increased (1.6- and 2.0-fold) and ADP affinities decreased (1.4- and 1.9-fold), respectively. All AtAPC
isoforms generally exhibit lower maximal velocities (Vmax)
for ATP than for ADP transport (Table 3). Since the Vmax
is proportional to the amount of actively transporting carrier proteins enhanced Ca2+-dependent activation of the
APCs should be reflected by an identical increase in the
Vmax of both, ADP and ATP transport. However, addition
of extra Ca2+ caused only a moderate increase (by approximately 1.2- to 1.5-fold) in maximal ADP Vmax but
stimulated the respective ATP Vmax (2.0- to 2.5-fold) of all
three APCs to a greater extend.

ATP transport stimulation by Ca2+ does not involve the
N-terminal domain

We choose AtAPC2 for a more detailed analysis of the
proposed ATP-Ca transport because ATP uptake of this
transporter was markedly stimulated by Ca2+and particularly because Ca2+ stimulation was only slightly affected
by Mg2+ presence (Fig. 2). To investigate ATP-Ca transport disconnected from possible Ca2+-dependent carrier
activation we generated an AtAPC2 mutant protein lacking the predicted N-terminal domain (Additional file 4:
Figure S4A and B). ATP uptake measurements verified
that truncated AtAPC2 is functional (Additional file 4:
Figure S4C), however, the uptake rates were slightly lower
than those of the full-length protein.
Determination of Ca2+ impact on transport activity
showed that ATP/Pi exchange via the mutated carrier

Table 3 Effects of 200 μM calcium on KM-Values of adenine nucleotide transport
Exchange

AtAPC1
KM

AtAPC2
Vmax

KM

AtAPC3
Vmax

KM

Vmax

ATP/Pi

68 (6)

201(±14)

95 (±12)

212 (±24)

113 (±17)

282 (±20)

ATP/Pi + Ca2+

61 (±6)

398 (±30)

59 (±6)

523 (±48)

58 (±8)

646 (±88)

ADP/ATP

180 (±15)

2078 (±315)

374 (±40)

778 (±82)

72 (±5)

770 (±90)

ADP/ATP + Ca2+

178 (±15)

2455 (±357)

508 (±94)

1169 (±126)

140 (±12)

1025 (±140)

Transport was performed with rising ATP or ADP concentrations and allowed for 2.5 min. KM- values are given in μM and Vmax in nmol mg protein-1 h-1. Data
represent the mean of at least three independent experiments. Standard errors are given in brackets

Lorenz et al. BMC Plant Biology (2015) 15:238

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Fig. 2 Ca2+-impact on ATP transport via AtAPC1-3. Effect of rising Ca2+-concentrations (0–500 μM) on transport mediated by recombinant AtAPC1
(a, b), AtAPC2 (c, d) and AtAPC3 (e, f). Transport of 50 μM [α32P]-ATP was conducted in absence (black rhombs) and presence of supplemental
MgCl2 (gray rhombs). Transport was allowed for 5 min and is given in nmol mg protein−1 h−1. Ca2+-dependent stimulation of ATP/Pi heteroexchanges (a, c, e) and ATP/ATP homo-exchanges (b, d, f). Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal
accumulation of ATP and the corresponding rates were unaffected by MgCl2 addition. Data represent mean values of three independent replicates,
standard errors are given

was considerably stimulated by increasing Ca2+ concentrations (~3-fold). Moreover, the degree of Ca2+-dependent
stimulation and the general course of the corresponding transport basically resembled that of the full-length
protein (Fig. 3, black squares). Investigation of ADP
uptake into ATP loaded liposomes revealed slight
transport stimulation of the full-length protein by low
Ca2+ concentrations (~35 % at 50 to 100 μM Ca2+),
which approached saturation at higher concentrations
(+60 %) (Fig. 3a, gray circles), whereas the corresponding transport of the truncated carrier version remained

rather unaffected by moderate Ca2+ concentrations
(+/− 10 % until 200 μM Ca2+) and became stimulated only
at higher Ca2+ concentrations (+50 %) (Fig. 3b, gray circles).
Although slight differences in the Ca2+-impact are detectable, the higher influence of Ca2+ on ATP than on
ADP import is apparently independent of the presence
or absence of the N-terminal domain. This result verifies
that the observed Ca2+-dependent ATP transport stimulation does not primarily result from carrier activation
and might rather be caused by increased ATP-Ca formation and substrate availability.


Lorenz et al. BMC Plant Biology (2015) 15:238

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Fig. 3 Ca2+-impact on ATP and ADP transport of full-length and N-terminally truncated AtAPC2. Transport via recombinant AtAPC2 (a) and via the
mutated version lacking its N-terminal domain (b). Import of [α32P]-ATP into Pi loaded proteoliposomes (black squares) and of [α32P]-ADP into ATP
loaded vesicles (gray circles) was allowed for 5 min. Transport without CaCl2 was set to 100 % and transport in presence of rising concentrations of
externally added CaCl2 (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and ADP/ATP uptake minus the respective control
(non-loaded vesicles) of three independent replicates. Standard errors are given

ATP but not ADP import of AtAPC2 requires the presence
of divalent cations

Because full-length AtAPC2 already exhibits basic ATP/
Pi exchange activity without extra Ca2+ addition and particularly because ADP uptake becomes not highly stimulated by rising Ca2+-concentrations (Fig. 3), it might be
assumed that the majority of reconstituted carriers is
already opened/activated due to contaminating Ca2+.
The cation chelator EGTA efficiently chelates Ca2+ (with
significant higher affinity than to Mg2+) and accordingly
should remove residual Ca2+ from the medium. We thus
used addition of EGTA to the transport medium to investigate whether and how Ca2+ depletion affects carrier activities. ATP/Pi exchange of full-length AtAPC2 becomes
significantly reduced by addition of 10 μM EGTA and further increase of its concentration causes total inhibition
(Fig. 4a, black squares). Interestingly, a similar inhibitory
effect was also observed for the truncated carrier version
(Fig. 4b, black squares). Given that the N-terminal domain
forms a lid that virtually closes the translocation pathway

when free Ca2+ is missing, efficient Ca2+-removal should
impede transport activity of AtAPC2 but not of the “uncapped” mutant. Moreover, ADP/ATP exchange of both,
full-length and truncated, AtAPC2 variants remained
more or less unaltered by EGTA addition (Fig. 4a and b,

gray circles). Accordingly, Ca2+ removal from the medium
did not cause inhibition of the overall transport capacity
by deactivation of the reconstituted carrier.
Interestingly, transport via AtAPC2 was not only
blocked by EGTA but also by the divalent cation chelator EDTA. Moreover, activity of the EGTA-inhibited
carrier could be fully restored by either Ca2+ or Mg2+
(Fig. 5). However, when compared to Ca2+ higher concentrations of Mg2+ are required for transport reactivation/stimulation.
So far we cannot explain explicitly why solely ATP
transport, but not general carrier activity, becomes inhibited by EGTA. It is imaginable that full-length AtAPC2
proteins are primarily or exclusively inserted in an insideout orientation, exposing the N-terminal domain to the

Fig. 4 Impact of rising EGTA concentrations on adenine nucleotide transport of full-length and N-terminally truncated AtAPC2. Transport via
recombinant AtAPC2 (a) and via the mutated version lacking its N-terminal domain (b). Import of [α32P]-ATP into Pi loaded proteoliposomes (black
squares) and of [α32P]-ADP into ATP loaded vesicles (gray circles) was allowed for 5 min. Transport without EGTA was set to 100 % and transport
in presence of rising concentrations of externally added EGTA (0 - 500 μM) was calculated accordingly. Data represent net values of ATP/Pi and
ADP/ATP import minus the respective control (non-loaded vesicles) of three independent replicates. Standard errors are given


Lorenz et al. BMC Plant Biology (2015) 15:238

Plant APC2 can mediate Ca2+-transport in vitro

EGTA pretreated
& reactivation

450
400

Page 9 of 16

337

no
pretreatment

350

302

Import [%]

300
250
200
150
110

100

111

100

71

58

40
5

8

EDTA

EGTA

50

CaCl2 (1.0)

CaCl2 (0.2)

MgCl2 (2.0)

MgCl2 (0.5)

MgCl 2 (0.2)

CaCl 2 (1.0)

MgCl 2 (0.5)

Control

0

Fig. 5 EGTA-inhibition of ATP transport via AtAPC2 and reactivation
by MgCl2 and CaCl2. Transport of 50 μM [α32P]-ATP into Pi loaded
vesicles in absence of EGTA, MgCl2 and CaCl2 was set to 100 %
(control; red line). Transport in presence of 200 μM EGTA, 200 μM

EDTA and the given concentrations (in mM) of MgCl2 or CaCl2 was
calculated accordingly. Generally transport was allowed for 10 min.
However, EGTA-inhibited transport was reactivated after 10 min of
uptake by subsequent addition of MgCl2 or CaCl2 and transport was
again allowed for 10 min (EGTA pretreatment and reactivation; light
gray bars). Data represent net values (ATP import in exchange with
Pi minus background values of non-loaded proteoliposomes) and
are the mean of three independent experiments. Standard errors
are indicated

liposomal interior. This orientation would clearly hinder
EGTA access to the regulatory sites (EF-Hands). However,
AtAPC2-proteoliposomes loaded with Pi and 200 μM
EGTA were still capable for ATP import (78 % of the corresponding EGTA-unaffected transport) (Additional file 5:
Figure S5). Moreover, inhibition of ATP uptake into these
EGTA-loaded liposomes by external EGTA as well as its
(re)activation by 500 μM external Ca2+ were nearly identical when compared to standard AtAPC2-proteoliposomes
lacking internal EGTA (Additional file 5: Figure S5).
Together, the obtained results indicate that ATP transport but not ADP or Pi transport of AtAPC2 essentially
requires the presence of divalent cations and this requirement is independent of the N-terminal domain and
thus not connected to carrier activation.

The observed Ca2+ and EGTA effects on AtAPC2 activity led us to the conclusion that Ca2+ might act as an
important co-substrate in ATP transport. To verify the
proposed capacity of AtAPC2 for ATP-Ca transport in
the liposomal system we performed uptake studies with
20 μM [45Ca] and 100 μM non-labeled ATP. Preliminary
analyses revealed that the read-out of the import rates
was hampered due to the high degree of nonspecific
[45Ca]-interaction with the phospholipids at the liposomal

surface (causing high radioactive background values).
However, reduction of these non-specific background
counts by removal of the vast majority of [45Ca] from the
liposomal surface was achieved by additional EGTA treatment of the vesicles subsequent to the uptake measurements (prior to vacuum filtration and washing). The
correspondingly modified transport assay allowed determination of small but significant time dependent Ca2+ uptake by full-length and truncated AtAPC2.
Ca2+ uptake into Pi loaded vesicles (Fig. 6a and b,
black rhombs) always exceeded the corresponding rates
obtained with non-loaded proteoliposomes (Fig. 6a and
b, gray squares) indicating that Ca2+ accumulation is directly connected to the antiport activity of the carrier.
The full-length protein exhibits higher Ca2+ transport
rates and also the back-ground values of the non-loaded
vesicles are enhanced when compared to the truncated
version (compare Fig. 6a and b). So far it cannot be discriminated whether - albeit EGTA treatment - a certain
amount of Ca2+ still binds to the N-terminal domain of
recombinant AtAPC2 or/and the functionality of the
truncated protein is generally slightly impaired.
Lastly, we analyzed effects of Mg2+ on Ca2+ import via
recombinant AtAPC2. For this, Pi loaded and nonloaded AtAPC2 proteoliposomes were incubated in
transport medium containing 20 μM [45Ca], 100 μM
non-labeled ATP and increasing concentrations of Mg2+.
[45Ca] import into phosphate loaded vesicles became significantly reduced by Mg2+ whereas the corresponding
rates of the non-loaded vesicles remained more or less
unaffected by Mg2+ addition (Fig. 6c). Quite high
amounts of Mg2+ (200 μM) are required to cause approximately half maximal transport inhibition whereas
25-fold excess of Mg2+ completely blocks Ca2+ uptake.
Because of the generally low [45Ca] transport rates of the
truncated AtAPC2 reliable interpretation of the corresponding results obtained with this protein is complicated. Nevertheless, the tendency of Mg2+ impact on
Ca2+ uptake generally resembles that of the full-length
protein (Additional file 6: Figure S6). The obtained
data suggest that Mg2+ competes with Ca2+ during

ATP complex formation and thereby can reduce ATPCa availability and hence Ca2+-import via the reconstituted carrier.


Ca2+ import [nmol/mg protein]

Lorenz et al. BMC Plant Biology (2015) 15:238

4.5

Ca2+ import [nmol/mg protein]

Fig. 6 Determination of Ca2+ transport via AtAPC2. Time dependent
uptake of [45Ca] via full-length AtAPC2 (a) and via N-terminally
truncated AtAPC2 (b) reconstituted into Pi (black rhombs) and
non-loaded liposomes (gray squares). (c) Effects of rising MgCl2
concentrations on [45Ca] transport into Pi loaded (dark gray bars)
and non-loaded (light gray bars) AtAPC2 proteoliposomes. Transport
media contained 20 μM [45Ca] and were additionally supplemented
with 100 μM non-labeled ATP and the indicated concentrations of
MgCl2. For determination of the Mg2+-effects on Ca2+ transport
via AtAPC2 uptake was allowed for 10 min (given as nmol mg
protein−1 h−1). Data represent mean values of three independent
replicates. Standard errors are indicated

A

4
3.5
3
2.5

2
1.5
1
0.5
0
0
2.5

5

10
15
Time [min]

20

B

2
1.5
1
0.5
0
0

Ca2+ import [nmol mg protein-1h-1]

Page 10 of 16

16

5

10
15
Time [min]

20

C

14
12
10
8
6
4
2
0
0 µM

50 µM
200 µM
500 µM
MgCl2 concentration

Discussion
Transport capacities of plant APCs allow energy exchange
as well as net adenine nucleotide provision

Diverse biological conditions, such as ATP-loading during
mitochondrial biogenesis or physiological and environmental changes, require modulation of the mitochondrial
adenine nucleotide pool size [9, 17, 18, 46]. During the
past decades net influx or efflux of adenine nucleotides
into or out of the organelle as well as the involved
carriers have been well studied in mammals and yeast
[9, 11, 12, 14–18, 46]. However, much less is known
about these processes in plants.
It is quite obvious that also plant mitochondria have
to adapt the adenine nucleotide concentration in the
mitochondrial matrix in accordance to the respective
metabolic demands. Already in the 1970s isolated corn
and cauliflower mitochondria were shown to exhibit
(carboxy)atractyloside insensitive (AAC independent)
uptake of adenine nucleotides [26–28]. In the beginning,
net import of ADP into plant mitochondria was identified to occur via exchange with Pi [31]. Later on, ADP
transport was shown to be influenced by Mg2+ and Ca2+
and it was suggested that exogenous rather than endogenous Pi drives net ADP uptake [29]. These inconsistencies might be due to the fact that mitochondria
harbor various carriers and enzymes directly or indirectly involved in adenine nucleotide transport and metabolism and that these proteins are differently affected
by the respective test conditions and metabolic states of
the organelle.
Arabidopsis thaliana encodes three MCF proteins
(AtAPC1-3) that represent promising candidates for net
adenine nucleotide transport. First of all, AtAPC1-3 exhibit significant amino acid similarities to APCs from
animals or yeast and contain the characteristic Nterminal domain with EF-hand motifs (Additional file 7:
Figure S7 and Additional file 8: Figure S8). Secondly,
these proteins can compensate the growth defect of
yeast Δsal1p mutants inhibited in AAC mediated transport [32]. Thirdly, transport assays performed in this
work with the reconstituted, recombinant carriers revealed that AtAPC1-3 act in a strict antiport mode

Lorenz et al. BMC Plant Biology (2015) 15:238

(Fig. 1, Additional file 2: Figure S2); they can catalyze
homo-exchanges of ATP and ADP as well as ATP/ADP
hetero-exchange but most importantly also ATP and
ADP hetero-exchange with Pi in vitro (Fig. 1, Additional
file 2: Figure S2, Tables 1, and 3). The latter capacity was
also shown recently in a study by Palmieri and coworkers that was published while this manuscript was in
revision [47]. Based on the in vitro characteristics
growth-restoration in the yeast complementation assay
by the three AtAPC isoforms [32] can be attributed to
their capacity for net adenine nucleotide supply (complementation of Sal1p activity) and/or for energy
provision (complementation of AAC activity).
Plant mitochondria possess a high affinity ADP uptake
system that is sensitive to AAC-specific inhibitors and a
low affinity ADP uptake system that apparently does not
involve AAC activity [30]. Biochemical characterization
of single isoforms suggest that AAC proteins mediate
the high affinity ADP transport [48] whereas APCs
catalyze or contribute to the low affinity ADP transport (Table 3) [47].
Interestingly, APC genes show more or less ubiquitous
expression with highest rates in growing tissues of enhanced mitochondrial propagation (Aramemnon, BAR
eFP browser; [49, 50]). The recent work by Palmieri and
coworkers showed that the promoter of Atapc1 exhibits
enhanced activity when compared to the remaining two
APC isoforms [47]. Moreover, expression of specific
isoforms (Aramemnon, GENEVESTIGATOR [49, 51])
is induced by growth-promoting plant steroids (brassinosteroides) or in response to abiotic stressors, like
hypoxia or phosphate limitation; conditions assumed to

be associated with altered mitochondrial metabolism/
respiration [45, 47, 52–54]. In future studies it will be
interesting to determine whether specific developmental stages or stress situations characterized by enhanced
or reduced APC expression correlate with the establishment or alteration of the mitochondrial adenine nucleotide pool.
Substrate preferences and impact of divalent cations on
transport

The fact that recombinant AtAPC3 and AtAPC1 apparently prefer homo-exchanges of ATP and ADP over
the corresponding hetero-exchanges with Pi (Fig. 1,
Additional file 2: Figure S2) might be indicative of
transport reduction due to unfavorable charge imbalances generated in the liposomes by the electrogenic
hetero-exchange. Similar to net ATP uptake by yeast
and mammalian mitochondria [11, 15, 16, 18] ATP
transport of AtAPC1 and AtAPC3 is markedly stimulated by Mg2+ (Fig. 1, Table 1). This stimulation occurs
during homo- and hetero-exchange and suggests that
AtAPC1 and AtAPC3 generally prefer ATP-Mg2− over

Page 11 of 16

ATP4− as import substrate independent of the generation of charge imbalances.
In contrast to AtAPC1 and AtAPC3, rates of homoand hetero-exchange of recombinant AtAPC2 are quite
similar (Fig. 1, Additional file 2: Figure S2) and ATP uptake was only slightly enhanced by Mg2+ addition (Fig. 1c
and d). These observations suggest that either a strong
preference of AtAPC2 for Pi as exchange substrate compensates possible negative effects of the charge imbalance of ATP/Pi (and ADP/Pi) hetero-exchange or that
hetero-exchange with Pi is not electrogenic at all. Interestingly, ATP transport of AtAPC2 was totally inhibited
by EGTA or EDTA and could be restored by Mg2+ or
Ca2+ (Fig. 5). This result strikingly argues for the requirement of divalent cations for ATP translocation.
Whether this is due to their function as co-substrate
and/or as effectors of the carrier protein cannot be unambiguously stated yet.
In contrast to our studies, Palmieri and coworkers investigated the capacity of ATP-Mg to act as export and

not as import substrate and under those conditions ATPMg transport is rather unfavorable when compared to
ATP [47]. Summarily, the current data therefore suggest
that the plant APCs possess different substrate preferences
at their exterior and interior side (Fig. 1, Table 1) [47].
Although Ca2+-dependent activity regulation of human and yeast APCs has been well known for a long
time, first insights into the mechanistic principle were
only gained recently. Sophisticated interaction studies
with human SCaMC1 suggest that in absence of Ca2+
the quite flexible N-terminal domain caps the transmembrane part whereas Ca2+-binding turns the Nterminal domain into a more rigid state which leads to
its dissociation and opening of the translocation pore
[21, 22]. Superimposition of the corresponding regions
in a structural alignment visualizes a high degree of
conservation among the N-terminal domains of plant
APCs and human SCaMC1 (Fig. 7). These structural
similarities as well as computer based docking analyses
(Additional file 8: Figure S8) suggest that the N-terminal
domains of the plant APCs also interact with four Ca2+ ions.
Moreover, amino acid sequence similarity to Sal1p and
human SCaMC isoforms suggest that plant APCs are likewise regulated by Ca2+ (Additional file 7: Figure S7, Fig. 7).
The fact that reconstituted APC isoforms from human
[12] and A. thaliana were already active without extra
Ca2+-addition led to the assumption that Ca2+ contaminations in the buffer media were sufficient for carrier activation. Because increase in Ca2+-concentrations resulted in
transport stimulation of all recombinant AtAPC isoforms
one might conclude that under the reconstitution conditions a mix of active and non-active carries occurs and
addition of Ca2+can thus activate additional carriers
(Fig. 2). However, the rates of Ca2+-stimulation were not


Lorenz et al. BMC Plant Biology (2015) 15:238

Page 12 of 16

Fig. 7 Structural alignment of the N-terminal domains of AtAPC1-3 and human SCaMC1. Three-dimensional homology models of AtAPC1
(residues 34–189, green), AtAPC2 (residues 38–194, yellow) and AtAPC3 (residues 35–189, orange). N-terminal domains were built using HHPred
server and Modeller based on the crystal structure of the Ca2+-binding N-terminal domain of human SCaMC1 (blue; PDB ID: 4N5X) in complex
with four calcium ions (gray spheres). The sequence alignment followed by a structural superimposition of the models was carried out using
PyMOL (version 1.3)

identical and varied depending on the kind of substrate
transported (Table 2).
Assuming that Ca2+ exclusively operates in carrier activation by displacement of the N-terminal domain from
the translocation pathway we would expect the same degree of (i) Ca2+-dependent transport stimulation, (ii)
Vmax increase (proportional to the amount of functional
carriers), and (iii) transport reduction by Ca2+-depletion
(with EGTA) independent of the exchanged substrates.
Moreover, truncation of the N-terminal domain should
cause constantly active carriers that are no longer influenced by Ca2+. However, the data obtained in this work
suggest that this is not the case. We therefore
hypothesize that in the in vitro system ATP-Ca acts as
substrate of the plant APCs and is even favored over
ATP-Mg or free ATP. By contrast, ADP-Ca seems to be
rather discriminated against when compared to free
ADP. Ca2+-induced alterations of the apparent transport
affinities most likely reflect these specific substrate preferences of the respective APC isoforms e.g. higher preference for ATP-Ca (when compared with the Mgcomplexed or free ATP) and lower preference of ADPCa (when compared to free ADP) (Table 3). Accordingly,
Ca2+ complexation of ATP enhances and that of ADP
reduces the amount of favored substrates and by this the
respective transport capacity of the reconstituted protein.
It is also imaginable that in the liposomal system, Ca2+ cotransport with ATP prevents charge accumulation of the
ATP/Pi hetero-exchange and with ADP3− (ADP-Ca1−) enhances the imbalance caused by the ADP/ATP heteroexchange. In addition, effects of EGTA, EDTA, Mg2+ and
Ca2+ on ATP transport inhibition, stimulation or reactivation suggest a competition between these cations during

complex formation and provide further evidences for
ATP-Ca as a potential in vitro substrate of recombinant
plant APCs (Table 2 and Figs. 3, 4, 5). We conclude that
the influence of Ca2+ on transport by the reconstituted

APCs is a consequence of diverse factors, such as substrate preferences, charge accumulation/compensation
and competition with Mg2+ during complex formation.
Transport characteristics obtained with AtAPC2 and
the N-terminally truncated version support the assumption that ATP-transport stimulation by Ca2+ is
not (or not exclusively) caused by activation of previously inactive (Ca2+-free) carriers. ATP transport of
both, the full-length carrier and the truncated version,
can be stimulated by Ca2+ and inhibited by EGTA
whereas ADP transport was not significantly affected
(Figs. 3 and 4). These results verify that solely ATP but
not ADP transport activity is highly dependent on the
presence of Ca2+ and that removal of this cation did
not cause carrier deactivation in general. The ineffectiveness of EGTA in the inhibition of total transport activity is surprising. The possibility that plant APCs are
generally not regulated in a Ca2+-dependent manner is
apparently not applicable. Important structural similarities of the plant, yeast and mammalian isoforms are
suggestive for a similar regulatory principle but most
importantly, a corresponding regulation could be demonstrated in the recent study by Palmieri and coworkers [47]. It remains unclear whether in our in vitro
system the functionality of the N-terminal domain of
the recombinant AtAPC2 is somehow impaired or its
affinity for Ca2+ is higher than that of EGTA. However,
the possibility that insight-out orientation of reconstituted
AtAPC2 and hence inaccessibility of the N-terminal domains caused ineffectiveness of EGTA in transport inhibition can be ruled out since proteoliposomes internally
loaded with EGTA were still capable to import ATP in exchange with Pi (Additional file 5: Figure S5).
The fact that external but not internal EGTA caused
inhibition of AtAPC2 mediated ATP import in exchange with Pi demonstrates that ATP but not Pi
transport requires the presence of Ca2+ (or divalent

Lorenz et al. BMC Plant Biology (2015) 15:238

cations). Moreover, this observation also demonstrates
that the chelator at the liposomal interior is apparently physically separated from Ca2+ at the exterior
(at least during the analyzed time span) which indicates that both, EGTA and Ca 2+, do not pass the lipid
barrier freely.
Notwithstanding or even because of the missing
Ca2+-dependent regulation, we were able to identify the
in vitro function of Ca2+ as co-substrate with the applied
system.
Although uptake studies with α[32P]-ATP provided
evidence for a possible ATP-Ca transport it would still
have been imaginable that Ca2+ stimulates transport of
unchelated ATP and impedes ATP-Mg transport in a different way. However, the specifically adapted uptake assay
using [45Ca] provided a direct proof that ATP-Ca is de
facto transported via reconstituted (Fig. 6). Time
dependent uptake of [45Ca] via AtAPC2 is tightly connected to its antiport activity because Pi loaded proteoliposomes accumulated higher amounts of [45Ca] than nonloaded vesicles. Competition experiments further verified
that ATP-Ca transport is favored over ATP-Mg transport
in vitro since quite high concentrations of Mg2+ are required to reduce ATP-transport associated Ca2+ uptake
(Fig. 6c, Additional file 6: Figure S6). When compared to
full-length AtAPC2 the N-terminally truncated carrier
shows reduced Ca2+ import capacity (Fig. 6b). Whether
absence of the N-terminal domain affects transport activity directly or rather indirectly (via impairments in refolding and membrane insertion) cannot be deduced from
these experiments.
Further studies with the reconstituted proteins as well
as with transgenic APC plants and isolated mitochondria
will be required to completely decipher, evaluate and
compare in vitro and in vivo characteristics of APC proteins. Moreover, it will be interesting to determine the

stoichiometry of the ATP and Ca2+ co-transport. Preliminary estimation suggests that these substrates are not
transported in a 1:1 stoichiometry. However, in this context it is important to mention that uptake assays had to
be adapted to make Ca2+ transport determination feasible
and furthermore that Ca2+ and Mg2+ contaminations of
the media have to be considered. Therefore, in future
studies we want to further optimize Ca2+-transport measurements in liposomes and intent to decipher the impact
of divalent cations on AtAPC1-3 function in vivo.

Page 13 of 16

that at least certain APCs can in principle accept ATPCa as substrate in vitro. However, the intriguing question
arises whether ATP-Ca formation and correspondingly
APC mediated Ca2+-transport can and will take place
under physiological conditions. Generally, ATP-Ca formation is a rather unlikely phenomenon in plant cells. The
concentration of free Ca2+ is usually low when compared
to Mg2+, which represents a dominating divalent cation
and also is Mg2+ favored over Ca2+ in ATP-complex formation. However, one could envision specific situations
that might support possible ATP-Ca formation in close
proximity to the carrier.
Although plant mitochondria contribute to Ca2+ storage, the majority of internal Ca2+ is probably transiently
fixed as amorphous phosphate precipitate and thus the
resting concentration of free Ca2+ in the matrix only
slightly exceeds that of the cytosol (200 nM vs. 100 nM)
[55–57]. Moreover, due to high Mg2+ concentrations
within plant mitochondria ATP is nearly completely
complexed with Mg2+, which argues against any potential ATP-Ca formation in the matrix [45]. Although
lower Mg2+ levels in the cytosol increase the accessibility
of free ATP, it is unclear whether conditions or microdomains of high Ca2+ availability at the mitochondrial surface might allow ATP-Ca formation [55, 58–63]. In the
liposomal system Ca2+ uptake via AtAPC2 was low and
completely blocked by 25-fold excess of Mg2+. If these

characteristics (the biochemical properties in combination with a high Mg2+ to Ca2+ ratio next to the carrier)
also represent the in vivo situation, ATP-Ca transport
via plant APCs is highly unlikely to occur.
Although, a direct role of plant APCs in ATP-Ca transport is therefore arguable, recent data suggest an indirect
function of a mammalian isoform in Ca2+ translocation.
SCaMC3 was shown to physically interact with the (low
affinity) Mitochondrial Calcium Uniporter (MCU) and
lack of SCaMC3 apparently decreases ATP and Ca2+ import into mitochondria [24, 64]. Accordingly, SCaMC3
was supposed to represent an important component of
the mitochondrial Ca2+ uptake system, a supercomplex
formed by channels and carriers in microdomains for enhanced Ca2+-sensitivity [64]. Whether certain plant APC
isoforms fulfill a function related to that described for
SCaMC3 is unclear, however, physical proximity to proteins involved in Ca2+ release might be advantageous to
guarantee fast Ca2+-dependent activation and response of
plant APCs.

Can ATP-Ca transport via plant APCs occur in vivo?

SCaMCs as well as yeast Sal1p seem to prefer ATP-Mg
whereas our initial studies indicate that at least one of
the AtAPC isoforms clearly favors ATP-Ca over both,
ATP-Mg and ATP, as import substrate in the liposomal
system. Due to the high structural similarity to ATP-Mg
it is - from a biochemical point of view - not surprising

Conclusions
Determination of the biochemical characteristics of three
putative APC isoforms from A. thaliana in the liposomal
system revealed that the recombinant carriers mediate
ATP, ADP and phosphate exchange. Accordingly, plant

mitochondria harbor a subset of carriers capable of net


Lorenz et al. BMC Plant Biology (2015) 15:238

adenine nucleotide translocation, however in contrast to
yeast and mammalian orthologs they show no high preference for ATP-Mg as import substrate. Surprisingly, we
instead obtained evidence for a possible ATP-Ca transport by the reconstituted plant APCs in the liposomal
context but it is arguable that physiological Mg2+ and
Ca2+ concentrations most likely prevent ATP-Ca formation and its subsequent transport in vivo. Although we
were not able to detect EF-hand based Ca2+-dependent
carrier regulation, this was shown recently to exist in
plant APCs [47]. Summarily, the current data suggest
that low Ca2+ concentrations regulate activity of plant
APCs via EF-hands of the N-terminal domain whereas
high Ca2+ concentrations can induce its own transport
as co-substrate of ATP in vitro. While this study deepens
our knowledge about mitochondrial net nucleotide
transport of plants it also gives rise to new intriguing
questions. In the future, it is important to investigate the
in vivo function of plant APCs and the impact of divalent cations on the corresponding transport.

Additional files
Additional file 1: Figure S1. Heterologously expressed AtAPC1-3
proteins accumulate in the inclusion body fraction of E. coli expression
cells. (A) SDS- PAGE of 5 μg and (B) Western-blot and immunodetection
of 0.5 μg of the inclusion bodies fraction from cells expressing AtAPC1
(lanes 1), AtAPC2 (lanes 2) and AtAPC3 (lanes 3). The Western-blot was
immuno-decorated with a monoclonal anti poly His IgG (Sigma, Taufkirchen,
Germany). M, prestained molecular weight marker (Thermo Fisher Scientific,

Schwerte, Germany) for estimation of the molecular masses (given in kDa)
of the recombinant proteins. (PDF 66 kb)
Additional file 2: Figure S2. Time dependent ADP transport via
AtAPC1-3. Transport of 50 μM [α32P]-ADP into Pi (A, C, E) and into ADP
(B, D, F) loaded proteoliposomes with reconstituted AtAPC1 (A, B), AtAPC2
(C, D) and AtAPC3 (E, F). Non-loaded liposomes (non-filled rhombs; negative
control) showed only marginal accumulation of radioactivity when
compared to proteoliposomes loaded with Pi or ADP (black rhombs).
Data represent mean values of three independent replicates, standard
errors are given. (PDF 82 kb)
Additional file 3: Figure S3. a. Determination of biochemical
parameters of ATP import into Pi loaded APC-proteoliposomes. Transport of
AtAPC1 (A, B), AtAPC2 (C, D) and AtAPC3 (E, F) was performed with rising
ATP concentrations in absence (A, C, E) or presence (B, D, F) of 200
μM CaCl2 and allowed for 2.5 min. Michaelis-Menten kinetics are the
mean of at least 3 replicates, SE are given. b. Determination of
biochemical parameters of ADP import into ATP loaded APC-proteoliposomes.
Transport of AtAPC1 (A, B), AtAPC2 (C, D) and AtAPC3 (E, F) was performed
with rising ADP concentrations in absence (A, C, E) or presence (B, D, F) of
200 μM CaCl2 and allowed for 2.5 min. Michaelis-Menten kinetics are the
mean of at least 3 replicates, SE are given. (PDF 126 kb)
Additional file 4: Figure S4. Heterologous expression and ATP
transport analysis of N- terminally truncated AtAPC2. (A) SDS-PAGE of
5μg and (B) Western-blot and immunodetection of 0.5 μg of the
inclusion bodies fraction from E. coli cells expressing the N-terminally
truncated (lanes 1). To enable detection of the molecular mass reduction
due to loss of the N-terminal extension the full-length protein was included
in this analysis (lanes 2). The Western-blot was immuno-decorated with a
monoclonal anti poly His IgG (Sigma, Taufkirchen, Germany). M, prestained
molecular weight marker (Thermo Fisher Scientific). (C) Time dependent

import of 50 μM [α32P]-ATP via N- terminally truncated AtAPC2 into ATP

Page 14 of 16

loaded (black rhombs), Pi loaded (gray circles) and non-loaded (non-filled
rhombs) liposomes. (PDF 156 kb)
Additional file 5: Figure S5. Impact of internal EGTA on ATP transport
via AtAPC2. Uptake of 50 μM [α32P]-ATP into proteoliposomes loaded
with Pi (black bars) or Pi plus 200 μM EGTA (light gray bars) was set to
100% (control). Inhibitory and stimulatory effects of externally added
EGTA (50 μM) and CaCl2 (500 μM) on the corresponding transport rates
were calculated accordingly. Reactivation of transport inhibited by
external EGTA was induced by addition of 500 μM CaCl2. Transport
(inhibition as well as activation) was allowed for 10 min. Data represent
net values (ATP/Pi exchange minus background values of non-loaded
proteoliposomes) and are the mean of at least three replicates. Standard
errors are indicated. (PDF 252 kb)
Additional file 6: Figure S6. Effects of rising MgCl2 concentrations on
[45Ca] transport via the N- terminally truncated AtAPC2. Transport of 20
μM [45Ca] into Pi loaded (dark gray bars) and non- loaded (light gray
bars) proteoliposomes was allowed for 10 min (given as nmol mg
protein-1 h-1). The transport medium was supplemented with 100 μM
non-labeled ATP and the indicated MgCl2 concentrations. Data represent
mean values of three independent replicates. Standard errors are indicated.
(PDF 102 kb)
Additional file 7: Figure S7. Alignment of APC proteins from different
organisms. Amino acid sequence alignment of APCs from A. thaliana
(AtAPC1-3 [GenBank:At5g61810; At5g51050; At5g07320]), S. cerevisiae
(Sal1p [GenBank: YNL083w]) and human (HsSCaMC1-3
[GenBank:SLC25A24; SLC25A25; SLC25A23] using ClustalW2 (http://

www.ebi.ac.uk). To allow easy detection of the N-terminal extension
mitochondrial AAC2 from S. cerevisiae (ScPET9 [GenBank:YBL030C]) was
included as a representative MCF protein. Shading of conserved amino
acid residues was performed with Boxshade at the Swiss EMBnet server
( Residues of the N-terminal
domains of AtAPC1-3 proposed to be involved in Ca2+-interaction are
highlighted by different colors. Residues predicted by Scanprosite (http://
prosite.expasy.org/scanprosite) are marked in green and by molecular
Ca2+ docking analyses with AutoDock vina (see also Additional file 8: Figure
S8) are marked in orange. Ca2+-interacting residues predicted by Scanprosite
and molecular docking studies are marked in yellow. EF-hands I and III
(orange boxes) exhibit lower support for Ca2+-interaction (Scanprosite) than
EF-hands II and IV (green boxes). (PDF 476 kb)
Additional file 8: Figure S8. Docking poses of Ca2+ ions within the
N-terminal domains of AtAPC1-3, interacting residues and structural
superimposition with human SCaMC1 (SLC25A24). Three-dimensional
homology models of the N-terminal domains of AtAPC1 (residues 34-189,
green), AtAPC2 (residues 38-194, yellow) and AtAPC3 (residues 35-189,
orange) were built using HHPred server and Modeller using the crystal
structure of the Ca2+-bound state of the N-terminal domain of human
SCaMC1 (blue) as template (PDB ID: 4N5X). The four EF-hand motifs
putatively involved in Ca2+ binding are marked in dark blue (A, C, E).
Docking poses of Ca2+ ions are shown for AtAPC1 N-term (A), AtAPC2
N-term (C) and AtAPC3 N-term (E) with residues putatively interacting
with Ca2+ marked in red. These residues were chosen either based on
docking or Scanprosite results ( For
the molecular docking analyses, Ca2+ ions and the N-terminal domains of
AtAPC1-3 were prepared using Autodock Tools 1.5.6. After determination of
the search space, the ions were docked into the structures using
Autodock vina. The best binding poses for Ca2+ were selected with respect

to the total energy and EF-hand positions. Structural superimposition of
AtAPC1 (B), AtAPC2 (D) and AtAPC3 (F) with SCaMC1 (blue) and Ca2+ ions
within this protein (blue spheres) was carried out using PyMOL (version 1.3).
(PDF 298 kb)
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
IH, HEN and UCV contributed to the conception of the study. AL, ML and IH
designed the experiments. AL and ML performed cloning and expression of
the carriers in the heterologous system. AL conducted and ML supervised
transport measurements and functional characterization of the carriers. SNW


Lorenz et al. BMC Plant Biology (2015) 15:238

performed amino acid sequence analyses and generated three-dimensional
models. AL, ML, SNW and IH collected the data and AL, HEN, UCV and IH
performed data interpretation. IH wrote the manuscript and was supported
by UCV and HEN. All authors read and approved the final manuscript.
Authors’ information
Not applicable
Availability of data and materials
Not applicable
Funding
The project was financially supported by the Deutsche Forschungsgemeinschaft
(Reinhard Koselleck-Grant). Work in the lab of UCV was supported by the
Deutsche Forschungsgemeinschaft (Center for Integrated Protein Science
Munich, CIPSM and VO656/5-1).
Author details
1

Cellular Physiology/Membrane Transport, University of Kaiserslautern, 67653
Kaiserslautern, Germany. 2Department of Biology I, Botany, LMU Munich,
Großhaderner Str. 2, D-82152 Planegg-Martinsried, Germany. 3Plant
Physiology, University of Kaiserslautern, 67653 Kaiserslautern, Germany.
Received: 25 June 2015 Accepted: 12 September 2015

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